Mechanism-based Suicide Inhibition

For all the enzyme inhibitors discussed so far, fluorine influences the mechanism of inhibition either by mimicking other functional groups or by influencing the conformation of the inhibitor by stereoelectronic effects. 

The best-known example of this type of inhibition is 5 -fluorouracil, a rather old cytostatic agent. Fluorouracil is first converted metabolically into the corresponding phosphodeoxyriboside. This, in turn, blocks DNA biosynthesis by inhibiting thy-midylate synthase, an enzyme which methylates deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP), one of the four building blocks of DNA [77]. 

The enzyme is irreversibly inhibited by the difluoromethyl analog of androstendione. The mechanism postulated for the inhibition involves oxidation of the difluoromethyl group and subsequent elimination of hydrofluoric acid. The resulting carbonyl fluoride binds covalently to a nucleophilic group of the enzyme, disabling it permanently [80]. 

Many fluorinated, mechanism-based inhibitors are amino acid derivatives [3, 81]. These target enzymes involved in amino acid metabolism, for example decarboxylases, transaminases or monoamine oxidases. 

The antiprotozoal drug Eflornithine is used to treat African sleeping sickness. 

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Figure 8.23. Mechanism-Based (Suicide) Inhibition. Monoamine oxidase, an enzyme important for neurotransmitter synthesis, requires the cofactor FAD (flavin adenine dinucleotide). AA -Dimethylpropargylamine inhibits monoamine oxidase by covalently modifying the flavin prosthetic group only after the inhibitor is first oxidized. The N-5 flavin adduct is stabilized by the addition of a proton.

During the past three decades, besides the rational design of hundreds of molecules that have been synthesized and tested as suicide substrates. It also has come to light that nature Itself has known about this mechanistic mode of enzyme Inhibition and provided us with several extremely potent mechanism-based suicide Inactivators. Below are a few selected examples to demonstrate the mode of action of these Inhibitors. 

The starting point for much of the work described in this article is the idea that quinone methides (QMs) are the electrophilic species that are generated from ortho-hydro-xybenzyl halides during the relatively selective modification of tryptophan residues in proteins. Therefore, a series of suicide substrates (a subtype of mechanism-based inhibitors) that produce quinone or quinonimine methides (QIMs) have been designed to inhibit enzymes. The concept of mechanism-based inhibitors was very appealing and has been widely applied. The present review will be focused on the inhibition of mammalian serine proteases and bacterial serine (3-lactamases by suicide inhibitors. These very different classes of enzymes have however an analogous step in their catalytic mechanism, the formation of an acyl-enzyme intermediate. Several studies have examined the possible use of quinone or quinonimine methides as the latent... 

Irreversible CYP inhibition can arise from different chemical mechanisms. However, a common initial step is the metabolic activation of a substrate into a reactive metabolite that is trapped within the active site of the CYP to form a tightly bound complex causing a long-lasting inactivation of enzyme activity. Enzymatic activity can be restored only through the new synthesis of the enzyme. For this reason, irreversible CYP inhibition is often referred to as mechanism-based inhibition , metabolite-based inhibition or suicide inhibition . 

When reactive metabolites are formed by metabolic activation, some of them can escape from the active site and bind to external protein residues or be trapped by reduced glutathione (GSH) or other nucleophiles. The remaining molecules that are not released from the active site will cause the suicide inhibition [7]. The ratio of the number of reactive molecules remaining in the active site and those escaping is a measure of the reactivity of the intermediates formed. The addition of scavengers or GSH to the incubation mixture does not affect and cannot prevent the CYP mechanism-based inhibition. However, GSH can reduce the extent of the nonspecific covalent binding to proteins by those reactive molecules that escape from the active site. In contrast, addition of substrates or inhibitors that compete for the same catalytic center usually results in reduction of the extent of inhibition. 

Allenic amino acids belong to the classical suicide substrates for the irreversible mechanism-based inhibition of enzymes [5], Among the different types of allenic substrates used for enzyme inhibition [128, 129], the deactivation of vitamin B6 (pyr-idoxal phosphate)-dependent decarboxylases by a-allenic a-amino acids plays an important role (Scheme 18.45). In analogy with the corresponding activity of other /3,y-unsaturated amino acids [102,130], it is assumed that the allenic amino acid 139 reacts with the decarboxylase 138 to furnish the imine 140, which is transformed into a Michael acceptor of type 141 by decarboxylation or deprotonation. Subsequent attack of a suitable nucleophilic group of the active site then leads to inhibition of the decarboxylase by irreversible formation of the adduct 142 [131,132]. 

A second type of CYP enzyme inhibition is mechanism-based inactivation (or suicide inactivation). In this type of inhibition, the effector compound (i.e., the in-... 

Irreversible inhibition with based-mechanism inhibitors (suicide-substrates)... 

Irreversible Inhibition with Mechanism-Based inhibitors (Suicide Substrates)... 

Linear furanocoumarins (psoralens) inhibit P450s as mechanism-based inactivators (suicide inhibitors). Thus, species that produce psoralens may have evolved C4H enzymes with enhanced tolerance to these compounds. Recombinant C4H from the psoralen-producing species R. graveolens showed markedly slower inhibition kinetics with psoralens, and possibly biologically significant tolerance, compared to C4H from a species that does not produce the compounds (H. tuberosus) ... 

Irreversible inhibitors combine or destroy a functional group on the enzyme so that it is no longer active. They often act by covalently modifying the enzyme. Thus a new enzyme needs to be synthesized. Examples of irreversible inhibitors include acetylsal-icyclic acid, which irreversibly inhibits cyclooxygenase in prostaglandin synthesis. Organophosphates (e.g., malathion, 8.10) irreversibly inhibit acetylcholinesterase. Suicide inhibitors (mechanism-based inactivators) are a special class of irreversible inhibitors. They are relatively unreactive until they bind to the active site of the enzyme, and then they inactivate the enzyme. 

L Hazen, LA Zupan, RH Weiss, DP Getman, RW Gross. Suicide inhibition of canine myocardial cytosolic calcium-independent phospholipase A2. Mechanism-based discrimination between calcium-dependent and independent phospholipases A2. J Biol... 

Mechanism-based inhibitors or suicide substrates seem to be particularly prevalent with CYP3A4. Such compounds are substrates for the enzyme, but metabolism is believed to form products that deactivate the enzyme. Several macrolide antibiotics, generally involving a tertiary amine function, are able to inhibit CYP3A4 in this manner (147,148). Erythromycin is one of the most widely used examples of this type of interaction, although there are other commonly prescribed agents that inactivate CYP3A4 (149-151), and a consideration of this phenomenon partially explains a number of interactions that are not readily explained by the conventional in vitro data (152). 

A final group of covalent small-molecule inhibitors of proteases are mechanism-based inhibitors. These inhibitors are enzyme-activated irreversible inhibitors, and they involve a two-hif mechanism that completely inhibits the protease. Some isocoumarins and -lactam derivatives have been shown to be mechanistic inhibitors of serine proteases. A classic example is the inhibition of elastase by several cephalosporin derivatives developed at Merck (Fig. 8). The catalytic serine attacks and opens the -lactam ring of the cephalosporin, which through various isomerization steps, allows for a Michael addition to the active site histidine and the formation of a stable enzyme-inhibitor complex (34). These mechanism-based inhibitors require an initial acylation event to take place before the irreversible inhibitory event. In this way, these small molecules have an analogous mechanism of inhibition to the naturally occurring serpins and a-2-macroglobin, which also act as suicide substrates. 

Mechanism-based inactivation of CYP450 (or suicide inhibition) occurs when a non-toxic drug is metabolised by CYP450 to generate a metabolite that can bind irreversibly with the enzyme. The mechanism of inhibition usually involves free-radical alkylation or acylation of the active site and results in destruction of enzyme activity. Examples of drugs that act in this way include the antibiotic chloramphenicol and the anticancer agent cyclophosphamide. 

The antibiotic chloramphenicol is oxidized by CYP monooxygenase to chloramphenicol oxamyl chloride formed by the oxidation of the dichloromethyl moiety of chloramphenicol followed by elimination of hydrochloric acid " (Figure 33.6). The reactive metabolite reacts with the e-amino group of a lysine residue in CYP and inhibits the enzymatic reaction progressively with time. This type of inhibition is a time-dependent inhibition or a mechanism-based inhibition or inactivation, and the substrate involved historically has been called a suicide substrate because the enzymatic reaction yields a reactive metabolite, which destroys the enzyme. ... 

A unique mechanism-based mode of action ( suicide inhibition ) for some fluoropharmaceuticals involves direct chemical reaction of a fluorinated substructure with the target protein. 

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